EGF-conditioned M1 macrophages Convey reduced inflammation into corneal endothelial cells through exosomes

Epidermal Growth Factor (EGF), a protein pivotal in cell proliferation and survival, has recently shown promise in alleviating inflammation. This study investigates EGF's impact on M1 macrophages, exploring its potential for anti-inflammatory and anti-vasculogenic interactions with corneal endothelial cells (CECs). Polarized M1 macrophages treated with EGF exhibited a suppression of gene expressions related to inflammatory and vasculogenic signals. The anti-inflammatory effects of EGF were observed in co-culture systems with human CECs (HCECs), showcasing its ability to alter macrophage phenotypes. Exosomes derived from EGF-treated M1 macrophages demonstrated enriched proteomic profiles related to immune system regulation and inflammation inhibition. When applied as eye drops in murine corneas, EGF-conditioned M1 macrophage-derived exosomes effectively reduced inflammation and increased M2-related ARG1 expression. This study highlights EGF's potential in mitigating inflammation in M1 macrophages and its delivery through exosomes to cultured HCECs and murine corneas, suggesting a novel therapeutic avenue for ocular surface anti-inflammatory treatments.


Introduction
The eye has long been recognized as one of the well-known immune-privileged organs [1].To maintain ocular immune privilege, particularly in the cornea, the absence of blood vessels is a normal feature [2].Additionally, the corneal endothelium serves as a barrier between the corneal stroma and the chamber, expressing surface molecules that inhibit complement activation, lymphocyte activation, and inflammation [3][4][5].However, the conjunctiva is equipped with mucosa-associated lymphoid tissue, known as conjunctiva-associated lymphoid tissue, making it easily susceptible to immunological activation [6].Immune cells also exist on the human cornea and they play a crucial role in mucosal immune responses to prevent infections and damage [7].Recent advancements, such as single-cell RNA sequencing, have identified various immune cell types in the corneal limbus, including CD8 + T cells, naïve T cells, double-negative T cells, as well as innate cells like macrophages, dendritic cells, monocytes, and basophils [8].
Among innate immune cells, macrophages form a diverse organ system across the body, acting as phagocytes to defend against pathogens and remove dead cells [9].CD11b + macrophages have been highlighted as the primary resident immune cells in the cornea, distributed from the central cornea to the peripheral limbus [10], constituting roughly 50% of all immune cells in the cornea [11].In healthy murine corneas, a CD11c − CD11b + population of bone marrow-derived cells, primarily representing monocytes/macrophages, is found in the posterior stroma [12].Corneal resident myeloid-lineage cells primarily recognize and mediate host responses to toll-like receptor (TLR) ligands through their pattern recognition receptors [13].For instance, exposure to the TLR9 ligand leads to macrophage accumulation on the corneal endothelium, forming multinucleated giant macrophages [14].This phenomenon, known as keratic precipitate, is a common sign of corneal stromal or endothelial inflammation in conditions like herpes simplex keratitis and uveitis [15].
Epidermal growth factor (EGF) is a well-known protein that stimulates cell proliferation, differentiation, and survival [16].Its role in tissue recovery after damage through EGF receptor (EGFR) signaling has been extensively studied, especially in skin [17].In the cornea, the distribution of EGFR in corneal epithelial cells has been reported [18].The presence of high basal levels of EGF in tear fluid suggests its importance in corneal epithelium restoration and homeostasis [19].However, the hypothetical role of EGF in the immune system remains controversial.EGFR activation has been linked to accelerated colitis and impaired disease recovery due to inhibition of interleukin (IL)-10 production [20].Conversely, heparin-binding EGF-like growth factor (HB-EGF) has been shown to decrease M1 polarization induced by lipopolysaccharide (LPS) and promote M2 polarization while protecting human fetal small intestinal epithelial FHs-74 cells [21].
Although corneal endothelial cells (CECs) are known to express EGFR ex vivo, the role of EGF in the interaction between macrophages and CECs has yet to be elucidated.This study aims to investigate whether EGF modulates M1 macrophages to facilitate antiinflammatory and anti-vasculogenic interactions with CECs.

EGF suppresses inflammatory and vasculogenic signals in M1 macrophages
To assess the influence of exogenous EGF on M1 macrophage inflammatory and vasculogenic activity, we treated M1 macrophages with human recombinant EGF at concentrations of 1 ng/mL and 10 ng/mL for 12 h.We observed reduced gene expression of inflammatory markers (IL6, IL1B, TNFA), phenotypical marker of macrophage (CD68) and vasculogenesis-related factors (VEGFA and VEGFC) (Fig. 2A).At the protein level, EGF treatment downregulated VEGF-A, VCAM-1, and IL-1β while upregulating the antiinflammatory cytokine IL-10.This coincided with increased intracellular EGF precursor protein levels in M1 cell lysates (Fig. 2B).ELISA analysis further confirmed reduced IL-6 secretion following EGF treatment (Fig. 2C).

EGF reduces inflammatory signals in corneal endothelial cells during co-culture with M1 macrophages
CECs are vital for maintaining corneal clarity and proper function [23].In the corneal stroma, macrophages are nearby and contribute to various functions, including immune responses and wound healing.This geometric arrangement enables them to contribute to innate immune responses, facilitate efficient wound healing, and uphold vessel homeostasis under steady-state conditions [12,24].On the contrary, macrophages have been implicated in mediating corneal endothelial rejection following corneal transplantation [25].We investigated EGF's anti-inflammatory effects in a co-culture system involving M1 macrophages and human CECs (HCECs) (Fig. 3A).

EGF modifies proteome composition in M1 macrophage-derived exosomes
Exosomes are small (40-160 nm) vesicles that facilitate cell-to-cell communication by delivering various signals [26].In a prior study, EGFRs were observed to be internalized in ex vivo CECs following EGF stimulation, suggesting a potential feedback inhibition mechanism for EGF functions in ocular surface cells [27].Hence, we conducted a proteome analysis on M1 macrophages to investigate if exosomes derived from EGF-conditioned M1 macrophages (EGF-M1 exo) carried inflammation-relieving signals compared to those from untreated M1 macrophages (M1 exo).Our aim was to screen proteins that might contribute to EGF-M1 exo's efficacy in alleviating inflammation.
We began by examining exosomes in both M0 and M1 macrophages (Fig. 4A) and then isolating and verifying exosomes from M1 macrophages based on their size and the presence of exosomal markers like Hsp70, CD63, and CD81 (Fig. 4B and C).EGF treatment  increased the exosomal expression of EGF precursor and the anti-inflammatory cytokine IL-10 in M1 macrophages (Fig. 4D).Next, we conducted a proteome analysis on M1-derived exosomes to identify compositional changes induced by EGF treatment.In total, we identified 2174 proteins in each exosome preparation.Of these, 2011 proteins were in M1 exo, and 1968 proteins were in EGF-M1 exo, with 1805 proteins common to both (Fig. 4E).
To understand the biological significance of these specific proteomes, we identified the top 25 proteins with the highest upregulation and down-regulation in relative abundance from the common proteome pool.Proteins more abundant in EGF-M1 exos included ras GTPase-activating protein 1, TNF-α-induced protein 8-like protein 2, and inactive rhomboid protein 2 (IRP2), which are known to negatively regulate apoptotic processes, T cell activation, and the inflammatory response to antigenic stimuli, respectively, within the Gene Ontology (GO) biological process category (Fig. 4F).In contrast, proteins down-regulated by EGF in exosomes included CD151 antigen, elongation factor 1-delta, and ribonuclease T2, which are known to play roles in T cell proliferation, positive regulation of I-kappaB kinase/NF-kappaB signaling, and the innate immune response, respectively (Fig. 4G).
We also conducted pathway analysis for proteins unique to M1 exo (206 proteins) and EGF-M1 exo (163 proteins) using Reactome pathway analysis.Interestingly, the heme degradation pathway, known for its role in regulating TNF-α and suppressing LPS-induced IL-6 production [24], was most highly involved in EGF-M1 exo's unique proteome pathways (Fig. S2).
Furthermore, we conducted a screening among whole exosomal proteome focused on proteins associated with immunological functions, utilizing categories like biological processes in GO, Wiki pathways, and Reactome pathways.In M1 exo-specific proteome, proteins including interferon regulatory factor 3, tyrosine-protein phosphatase non-receptor type 11, interferon regulatory factor 2binding protein 2, TNF, and VCAM-1, which are involved in immune system activation, were found (Fig. S3A).In EGF-M1 exo-specific proteome, collagen alpha-2(I) chain, superoxide dismutase, transforming growth factor beta-1, and signal transducer and activator of transcription 2, which have role in negative regulation of inflammatory-and immunologic signals, were found (Fig. S3B).
Among the targets where changes in mRNA levels were confirmed in both M1 macrophages and HCECs in Figs. 2 and 3, the relative abundance of IL-1β and ICAM-1 in the exosomal proteome pool decreased with EGF treatment, showing ratios of 0.30 (EGF-M1 exo/M1 exo) and 0.71, respectively.VCAM-1 was notably absent in EGF-M1 exo but exhibited high presence in M1 exo.VEGF-A, PDGFA, and MMP-2 were highly prevalent in both M1 exo and EGF-M1 exo, making the difference in their existence levels equivocal.

EGF-conditioned M1 macrophage exosome eye drops alleviate inflammation and vasculogenic signals in murine cornea
We further evaluated the effects of M1-derived exosomes on murine eyes.First, we investigated the absorption of exosome eye drops on the ocular surface and within the eye.After applying M1 exo eye drops three times over 24 h, we detected fluorescence from the applied M1 exos in various ocular areas, including the cornea, ciliary body, retina, and eyelid (Fig. 6).Notably, exosomes were successfully delivered into the eyelid's glandular system.
Next, we compared the effects of M1 exo and EGF-M1 exo on inflammatory signals in murine eyes.Using C57BL/6 mice, we removed the corneal epithelium and administered M1 exo and EGF-M1 exo eye drops a total of eight times.Subsequently, we excised the corneal buttons for analysis (Fig. 7A).After treatment with M1 exo eye drops, we observed cells expressing F4/80, CD68, or CD86 on the corneal endothelium, indicating keratic precipitate formation (Fig. 7B).M1 exo eye drops increased the gene expression of inflammatory and vasculogenic factors (IL1B, IL6, VCAM1, and VEGFA) in the excised corneal tissues.In contrast, EGF-M1 exo eye drops did not induce the gene expression of IL6 and VCAM1 (Fig. 7C).
Both M1 exo and EGF-M1 exo eye drops elevated the gene expression of macrophage markers CD14, CD11B, and CD11C in corneal tissues.However, gene expressions of CD68, CD86, and ADGRE1 which is associated with F4/80, were not upregulated by EGF-M1 exo eye drops.Intriguingly, the ARG1 gene expression, linked to arginase-1 and indicative of anti-inflammatory M2 macrophages, significantly increased in response to EGF-M1 exo eye drops compared to M1 exo eye drops (Fig. 7C).

Discussion
Our results demonstrate the anti-inflammatory and anti-vasculogenic effects of EGF on M1 macrophages and its impact on HCECs and the murine cornea.Specifically, conditioning M1-polarized macrophages with EGF suppresses their intracellular inflammatory and vasculogenic signals and alters the immunological composition of exosomes in M1 macrophages.Consequently, while the direct application of EGF to HCECs during co-culture with M1 macrophages yielded unsatisfactory results, delivering EGF-conditioned M1derived exosomes into HCECs significantly attenuated inflammatory and vasculogenic signals.Fortunately, the effect of EGF-M1 exo eye drops partially differed from the effect of M1 exo eye drops in the murine cornea.This difference was represented by the attenuation of IL6 and VCAM1 gene expression and activation of ARG1 gene expression.
In induced M1 macrophages from THP-1 monocytes, stimulating EGFR of M1 macrophages using EGF, a prototype ligand of EGFR, downregulated inflammatory and vasculogenic signals.While EGFR is present on human macrophages [28], its role in macrophages has mainly been studied in cancer research.However, EGFR has been found on rabbit peripheral monocytes and macrophages, where it promoted cell proliferation and migration [29].Although we did not investigate the signal directly stimulating EGFR in M1 cells, it is likely that EGF exerts anti-inflammatory mechanisms in M1 cells derived from THP-1 cells.
Unfortunately, the direct application of EGF in HCECs did not significantly downregulate IL6 gene expression.In contrast, EGF-M1 exo treatment in HCECs effectively suppressed IL6 gene expression compared to M1 exo treatment.This discrepancy in the effect of EGF suggests that EGFR-mediated signaling may be easily compensated for by a negative feedback mechanism.Indeed, EGFR internalization in ex vivo rat CECs occurred rapidly within 15-30 min after EGF stimulation [27].
While the application of EGF-M1 exosomes led to the suppression of IL1B, IL6, VCAM1, ICAM1, VEGFA, PDGFA, MMP2, and MMP9 expressions, the pre-conditioning concentrations of EGF required for inhibiting inflammatory or vasculogenic signals varied across targets.The reason for this variation remains unknown, but we hypothesize that compensatory signals triggered by EGF may differ among the targeted molecules.Macrophages are known to closely associate with adjacent tissue cells in response to reciprocal growth factor signals, including EGF, in various tissues, even in a steady state [30].Considering the importance of growth factors in maintaining homeostatic interactions between macrophages and tissue cells, the optimal EGF concentration may be contingent upon the microenvironmental conditions of inflammation.Further investigation into this issue is warranted for future studies.Following EGF   conditioning of M1 macrophages, we observed an increased amount of IRP2 among exosomal proteomes.We consider IRP2 as a possible representative of the EGF-related anti-inflammatory mechanism in M1 macrophages, as IRP2 is involved in the "CD163 mediating an anti-inflammatory response" pathway and interacts with and regulates a disintegrin and metalloprotease-17 (ADAM17) protease [31].ADAM17 plays a crucial role in the proteolytic release and activation of several ligands of EGFR, as well as the IL-6 receptor [32].Analyzing changes in IRP2 and ADAM17 expression during EGFR stimulation or in EGF-conditioned macrophages could be an interesting topic for future research.
Topical instillation of eye drops made from chemicals often has limitations, such as the need for frequent application and low bioavailability.Exosome eye drops may offer a solution to these problems due to their high penetration capability through barriers.In this study, only three regular exogenous applications of exosome eye drops penetrated the epithelium-peeled cornea and were easily delivered to the ciliary body and retina.Similarly, salivary gland epithelial cell-derived exosome cargos were loaded with autoantigens Ro/SS-A, La/SS-B, and Smith ribonucleoprotein, which were then used to immunologically induce Sjogren's syndrome [33].
While M1 exo eye drops successfully induced inflammatory signals in mouse corneal tissue, unfortunately, EGF-M1 exo eye drops did not significantly ameliorate the upregulated expressions of inflammatory IL1B, macrophage markers including ADGRE1 and CD68, M1 marker CD86, and myeloid cell markers ITGAM and ITGAX, except IL6.Since EGF treatment in M1 macrophages did not induce trans-polarization into anti-inflammatory M2 macrophages (data not shown), it suggests that EGF preconditioning did not transdifferentiate M1 macrophages into other subtypes of macrophges.We hypothesized that EGF-M1 exo eye drops might also induce inflammatory signals in murine corneas given that EGF-M1 exo was also originally derived from M1 macrophages in fact.Zhao et al. reported that the activation of EGFR in macrophages using HB-EGF mediates negative feedback inhibition of M2 polarization in Raw 264.7 cells and peritoneal macrophages [34].On the contrary, HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation in macrophages derived from THP-1 human monocyte cell lines [21].Thus, the effect of EGFR stimulation may vary depending on the type of macrophages and the ligands of EGFR.While we were unable to discern the EGF-induced trans-differentiation of M1 macrophages into anti-inflammatory macrophages, the lack of upregulation of ADGRE1, CD68, and CD86 as well as the activation of ARG1 in murine corneal tissues after EGF-M1 exo eye drop application, suggests that EGF-conditioned macrophages might deliver signals capable of modulating the phenotype of M1 macrophages in vivo.This modulation appears to deactivate the M1 phenotype, particularly in vivo.
In murine corneal tissues, the application of EGF-M1 exo eye drops did not induce IL6 activation, in contrast to IL1B, which was activated even with EGF-M1 exo eye drops.Furthermore, IL6 suppression was observed in HCECs treated with EGF-M1 exo.However, in a co-culture system of HCECs and M1 macrophages, EGF treatment did not suppress IL6 gene expression in HCECs.Despite the traditional acceptance of IL-6 as a pro-inflammatory cytokine secreted by macrophages, it is known to have both pro-and antiinflammatory properties, depending on its classic versus trans-signaling [35].Therefore, caution is warranted when interpreting the suppression of the IL6 gene and IL-6 secretion by EGF in M1 cells, as it may not unequivocally induce anti-inflammation in corneal tissues or HCECs.
In conclusion, our findings demonstrate EGF's roles in ameliorating inflammation in M1 macrophages and its delivery through exosomes to cultured HCECs and murine corneas.This highlights that EGF's novel potential for anti-inflammatory therapies in the ocular surface.

Limitations of the study
We used the THP-1 human monocyte cell line to verify phenotypical alterations with EGF, which may not fully reflect the conditions of macrophages in the cornea.Additionally, we did not investigate the EGFR-dependent action of EGF on macrophages and HCECs, which limits our ability to provide a mechanistic explanation for the anti-inflammatory role of EGF on macrophages and its  In control group, the vehicle eye drops were applied in the unilateral eye after the corneal epithelial peeling.In exosome group, the M1 macrophage-derived exosome (M1 exo) eye drops and EGF-conditioned M1 macrophage-derived exosome (EGF-M1 exo) eye drops were applied in right and left eye, respectively, after the corneal epithelial peeling.The eye drops were applied 3 times a day and total 8 drops.(B) The immunofluorescene stain of pan-macrophage markers including F4/80 and CD68, and CD86 as a M1 macrophage marker in the excised subsequent effects on HCECs.The modifications in the expressions of different inflammatory and vasculogenic factors were predominantly confirmed at the mRNA levels rather than the protein levels.Additionally, the resulting alteration in inflammationtriggered vasculogenesis formations with EGF was not validated.The optimal concentration for preconditioning M1 macrophages with EGF, leading to a significant suppression of inflammatory and vasculogenic targets through exosomes, has not been established.Nonetheless, there have been no previous studies investigating the novel role of EGF beyond epithelial wound healing on the ocular surface.Future experiments to confirm the possible anti-inflammatory target of EGF in corneal macrophages are necessary to understand EGF's specific role in the corneal innate immune system.

Study approval
We obtained approval from the Institutional Review Boards at Chung-Ang University Hospital (Approval No. 2106-006-465) for using cadaveric ocular tissues in corneal endothelial culture, following the principles of the Declaration of Helsinki.The Institutional Animal Care and Use Committee of Chung-Ang University approved the experimental protocol (IACUC approval No. 2020-00127), aligning with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Culture of human corneal endothelial cells
We followed a previously established protocol for culturing human corneal endothelial cells (HCECs) [37].Human corneal tissues without ocular diseases were obtained for transplantation and stored at 4 • C in Optisol-GS storage medium (Bausch & Lomb, Rochester, NY, USA).After excising the central 8-mm round cornea for transplantation, we utilized the peripheral corneal tissues for HCEC culture.These corneal tissues were thoroughly washed with phosphate-buffered saline (PBS, WelGENE) containing antibiotics (penicillin/streptomycin). To isolate HCECs, we carefully removed Descemet's membrane and corneal endothelium, followed by digestion with 1 mg/ml collagenase A (#07434, STEMCELL Technologies Inc, Vancouver, Canada) at 37 • C for 2 h.The detached HCECs were placed in corneal endothelial cell growth medium (Opti-MEM™-I, #31985070, Gibco, NY, USA) supplemented with various components.HCECs were collected after centrifugation at 1200 rpm for 5 min and plated on FNC-coated tissue culture dishes.Cells were cultured at 37 • C in a humidified 5% CO 2 incubator and sub-cultured when reaching 80-90% confluency using TrypLE Express (Gibco).

Co-culture of M1 macrophages and HCECs
HCECs were seeded in 6 well-plates at a density of 1.5 × 10 5 cells/well and cultured for 24 h.Co-culture were conducted using transwell inserts with a pore size of 0.4 μm (SPL Life Sciences, Pocheon-si, South Korea) placed into 6-well plates.M1 macrophages were resuspended in RPMI 1640 at density of 1 × 10 5 cells/insert and then treated with EGF.The co-cultures of M1 macrophages and HCECs were incubated undisturbed at 37 • C in a humidified 5 % CO 2 incubator for 24 h.RNA was subsequently extracted from HCECs (6-well plate) for analysis.

S.J. Lee et al.
and heat shock protein 70 (Hsp70) were assessed using western blotting, as described in the Western blot analysis section.
The size distribution and concentration of purified exosomes were measured using nanoparticle tracking analysis with a Malvern NanoSight NS300 (Malvern Instruments Company, Malvern, England) and the corresponding software NanoSight NTA 3.4 Analytical.

Exosome staining and tracking
To label the exosomes, we used the PKH26 red fluorescent cell linker dye (#MINI26, Sigma-Aldrich) following the manufacturer's instructions.The exosomes were stained by adding 1 μL of PKH26 dye to 200 μL of Diluent C fluid from the kit and incubating them for 5 min at room temperature (RT).To halt the labeling process, we added an equal volume of 10 % BSA and re-purified the exosomes using the total exosome isolation reagent precipitation method.The culture medium of HCECs was replaced with medium containing PKH26-labeled M1-derived exosomes.The cells were incubated with these exosomes for 16 h and then fixed with 4 % paraformaldehyde after washing with PBS.Subsequently, we labeled the cells with Wheat Germ Agglutinin (WGA; #W11261, Invitrogen) for 30 min at RT and stained the nuclei with 4′,6-diamidino-2-phenylindole (DAPI).The cells were analyzed using an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany).

Transmission electron microscope
For the observation of exosomes using transmission electron microscopy, the exosome pellet was initially fixed with 2.5 % glutaraldehyde and post-fixed with 2 % osmium tetroxide (OsO 4 ) for 1 h.Subsequently, the exosome pellet was dehydrated using a graded series of acetone and embedded in the Spurr Low Viscosity Embedding Kit (#EM0300, Sigma-Aldrich).Ultrathin sections, approximately 65 nm thick, were prepared using an EM UC7 ultramicrotome (Leica).These sections were then placed on a copper grid, stained with uranyl acetate and lead citrate, and examined using a TEM (HT7800; Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV.

Proteomic analysis of exosomes by mass spectrometry
We established the protein profiles of exosomes isolated from macrophages with and without preconditioning with EGF using Liquid Chromatography (LC)/Mass Spectrometry (MS), as previously described [38].In brief, we analyzed half of the digested sample through nano LC-MS/MS with a Waters NanoAcquity HPLC system connected to a ThermoFisher Q Exactive mass spectrometer.
Peptides were loaded onto a trapping column and eluted over a 75 μm analytical column at a flow rate of 350 nL/min, employing a 2-hr reverse phase gradient.Both columns were packed with Luna C18 resin (Phenomenex).The mass spectrometer operated in data-dependent mode, with the Orbitrap running at 70,000 FWHM and 17,500 FWHM for MS and MS/MS, respectively.We selected the fifteen most abundant ions for MS/MS analysis.
Proteins were identified using a software (Proteome Discoverer™, Thermo Fisher Scientific) and compared against the human SwissProt protein databases.To assess the up-regulation or down-regulation of proteome among commonly identified proteins in M1 exos and EGF-M1 exos, and to quantify the extent of change, we utilized the relative abundance ratio (EGF-M1 exo/M1 exo).The proteome abundance data was obtained from the normalized values shown in Proteome Discoverer™ software.To explore the activated pathways between M1 exo-specific proteome and EGF-M1 exo-specific proteome, Reactome pathway analysis (https://reactome.org) was employed.We represented the top 25 pathways, ranked by the p-value.Subsequently, we screened all individual exosomal proteins in M1 exos and EGF-M1 exos, identifying immunologically relevant proteins.The determination of immunologically relevance relied on functional categories, including biological process from GO, Wiki pathway, and Reactome pathway information provided in Proteome Discoverer™ software.A protein was considered immunologically relevant if the annotated words in each category were directly or indirectly associated with immunology, inflammation, cell survival/death, and the related signals.

Purification of total RNA and Real-Time qRT-PCR
Total RNA was extracted from cells and mouse cornea tissue using the NucleoZOL (#740404.200,Macherey-Nagel, Germany) according to the manufacturer's instructions and quantitated using NanoDrop™ One (Thermo Fisher Scientific).Single-stranded complementary DNA (cDNA) was synthesized from 1 μg of total RNA using RevertAid First Strand cDNA Synthesis Kit (#K1622, Thermo Fisher Scientific).Real-time RT-PCR was conducted using a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems™, CA, USA).Relative gene quantities were obtained using the comparative cycle threshold (Ct) method after normalization to 18S rRNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene.

Flow cytometry
For plate-differentiated THP-1 cells, 1 × 10 6 cells were added per tube.The cells were washed with cell staining buffer (1% BSA, 0.01% sodium azide in PBS) by centrifuging at 350×g for 5 min, and then we decanted the supernatant.The cells were blocked with 5 μL of human TruStain FcX (Fc receptor blocking solution; BioLegend) per 100 μL cell suspension for 10 min at RT, followed by a 20 min incubation in the dark and on ice, with the fluorochrome-conjugated anti-human antibodies (either anti-CD14 PE, anti-CD11b FITC, anti-CD11c APC or anti-CD14 PE, anti-CD86 APC, anti-CD163 FITC, anti-CD206 BV421).Following repeat of the wash step, and then resuspend cell pellet in 0.5 mL of cell staining buffer and add 5 μL of 7-AAD Viability staining solution (BioLegend) on ice in the dark.
The cells were analyzed on flow cytometer (FACSCanto, BD BioSciences, Mountain View, CA, USA).The data was analyzed using FlowJo software (BD, Ashland, OR, USA).

Animal experiment
Twenty 6-week-old BALB/c male mice (Orient Bio Inc., Seongnam, Gyeonggi-do, Korea) were used.The corneal epithelium was removed using a No. 15 blade before applying exosome eye drops.The control group received 5 μL of PBS in one eye, while the exosome group received 30 μg/5 μL of M1 exos diluted with PBS in the right eyes and 30 μg/5 μL of EGF (10 ng/mL)-M1 exos diluted with PBS in the left eyes.Each eye received a total of 8 applications (3 times/day).After sacrificing the mice, corneal buttons were excised for analysis.

Statistical analyses
Statistical analysis software (GraphPad Prism, ver. 10, GraphPad Software Inc., La Jolla, CA, USA) was used for statistical tests.To compare three or more groups, data were analyzed by the parametric ANOVA followed by Tuckey's post-hoc or non-parametric Kruskal-Wallis test followed by Dunn's post-hoc.To compare two groups, data were analyzed by the parametric Student's t-test or non-parametric Mann-Whitney U test.Data within graphs were presented as the mean ± standard error.Differences were considered statistically significant when P < 0.05.
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Fig. 4 .
Fig.4.Verification of M1 macrophage-derived exosomes and proteomic analysis in exosomes.(A) Representative transmission electron microscope photographs of multivesicular bodies (MVBs, arrows) and released exosomes from M1 macrophage (M1 exo).Scale bar: 500 nm.(B) Size and concentration of M1-derived exosomes evaluated by particle tracking analysis method.(C-D) The representative Western blot image for exosomal markers including Hsp70, CD63, CD81, and EGF precursor and IL-10 protein in M1 macrophage-derived exosomes with and without EGF treatment.The small gaps indicate skipped lanes from the same membrane.Full-length gels before cropping are noted in Fig.S5.Cropped gels are accompanied by full-length gels.(E) The number of proteins in each M1 exo and EGF-conditioned M1 macrophage-derived exosome (EGF-M1 exo) according to proteomic analysis of exosomes by mass spectrometry.(F-G) Most upregulated (F) or downregulated (G) 25 proteins among exosomal proteome in common from M1 exos and EGF-M1 exos based on the relative abundance of exosomal proteins.EGF, epidermal growth factor; IL-10, interleukin 10.S.J. Lee et al.
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Fig. 6 .
Fig. 6.The intraocular delivery of the exosome eye drops in corneal epithelium removed murine mouse model.The existence of PKH26 dye-labeled exosome verified by the immunofluorescence stain in various ocular areas, including the cornea, ciliary body, retina, and eyelid after 3 regular applications of M1 macrophage-derived eye drops (30 μg/5 μL per 1 drop) over 24 h.In the eyelid, the delivered exosomes from eye drops were delivered into the glandular system of the eyelid (arrow).Scar bars: 200 μm.

Fig. 7 .
Fig. 7.The alteration of inflammatory and vasculogenic signals corneal tissue after the application of eye drops made of EGF-conditioned M1 macrophages in corneal epithelium removed murine mouse model.(A) The scheme of animal experiments.The Balb/c mice were divided into control and exosome groups.In control group, the vehicle eye drops were applied in the unilateral eye after the corneal epithelial peeling.In exosome group, the M1 macrophage-derived exosome (M1 exo) eye drops and EGF-conditioned M1 macrophage-derived exosome (EGF-M1 exo) eye drops were applied in right and left eye, respectively, after the corneal epithelial peeling.The eye drops were applied 3 times a day and total 8 drops.(B) The immunofluorescene stain of pan-macrophage markers including F4/80 and CD68, and CD86 as a M1 macrophage marker in the excised and D are indicated in fig.S4 and fig.S5, respectively.